Two Phases of Mitogenic Signaling Unveil Roles for P53 and EGR1 in Elimination of Inconsistent Growth Signals

Molecular Cell Article

Two Phases of Mitogenic Signaling Unveil Roles for p53 and EGR1 in Elimination of Inconsistent Growth Signals

Yaara Zwang,1 Aldema Sas-Chen,1 Yotam Drier,2 Tal Shay,2 Roi Avraham,1 Mattia Lauriola,1 Efrat Shema,3 Efrat Lidor-Nili,3 Jasmine Jacob-Hirsch,4 Ninette Amariglio,4 Yiling Lu,5 Gordon B. Mills,5 Gideon Rechavi,4 Moshe Oren,3 Eytan Domany,2 and Yosef Yarden1,* 1Department of Biological Regulation 2Department of Physics of complex Systems 3Department of Molecular Cell Biology The Weizmann Institute of Science, Rehovot 76100, Israel 4Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel 5Department of System Biology, The University of Texas M.D. Anderson Cancer Center, Box 950, Houston, TX 77030, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.04.017

SUMMARY 6 hr later by the presence of nutrients and progression factors (Pledger et al., 1977; Stiles et al., 1979). A later report found Normal cells require continuous exposure to growth that continuous exposure to the platelet-derived growth factor factors in order to cross a restriction point and (PDGF) may be substituted by two pulses, separated by commit to cell-cycle progression. This can be re- a fixed-length interval (Jones and Kazlauskas, 2001). Based on placed by two short, appropriately spaced pulses this scenario, it was proposed that the first pulse primes of growth factors, where the first pulse primes a process, which is completed by the second pulse and enables a process, which is completed by the second pulse, R point transition (Kazlauskas, 2005). Our study investigated the dual-step process in mammary and enables restriction point crossing. Through inte- epithelial cells, stimulated by the epidermal growth factor gration of comprehensive proteomic and transcrip- (EGF). Like in fibroblasts, GF signaling promotes epithelial prolif- tomic analyses of each pulse, we identified three eration by regulating cyclins, cyclin-dependent kinases (CDKs), processes that regulate restriction point crossing: and CDK inhibitors (Stull et al., 2004). CDK-mediated inactivation (1) The first pulse induces essential metabolic en- of pRb facilitates release and activation of a group of transcription zymes and activates p53-dependent restraining factors (TFs), E2Fs, thus enabling progression from G1 to S phase processes. (2) The second pulse eliminates, via the (Chen et al., 2009). E2Fs are regulated by a bistable, switch-like PI3K/AKT pathway, the suppressive action of p53, mechanism essential for R point transition (Planas-Silva and as well as (3) sets an ERK-EGR1 threshold mecha- Weinberg, 1997; Yao et al., 2008). Following extracellular cues, nism, which digitizes graded external signals into c-MYC acts as an additional critical regulator of progression an all-or-none decision obligatory for S phase entry. through G1. Unlike transformed cells, which often harbor high expression of c-MYC, the abundance of this protein is tightly Together, our findings uncover two gating mecha- regulated in normal cells (Meyer and Penn, 2008). The expression nisms, which ensure that cells ignore fortuitous and stabilization of c-MYC cooperate with the bistable activation growth factors and undergo proliferation only in mode of E2F by inducing the expression of cyclins and by coop- response to consistent mitogenic signals. erating with E2F in a positive feedback loop (Leung et al., 2008). To unravel the molecular events that precede R point transition, we applied Kazlauskas’s two-pulse scenario to normal INTRODUCTION human mammary epithelial cells. Employing proteomic and transcriptomic analyses, we identified mechanisms that refute Growth factor (GF) signaling is continuously required during the mitogenic stimuli, unless they are consistent and appropriately G1 phase of the cell cycle, until cells cross a restriction (R) point, timed. Specifically, along with forward-driving processes, the after which cell-cycle progression becomes GF independent first pulse initiates also a restraining mechanism entailing p53 (Pardee, 1974). Quiescent fibroblasts typically require 12 hr to and a battery of antiproliferative genes. The second pulse progress through G1, until they enter the S phase (Stiles et al., engages a phosphoinositide 3-kinase (PI3K)-mediated mecha- 1979). R point crossing, however, occurs after prolonged (9 hr) nism that removes the p53-centered blockade. In addition, the exposure to GFs and precedes initiation of DNA synthesis. Early second pulse enhances extracellular signal-regulated kinase studies proposed that this interval comprises two phases: In the (ERK) signaling, in what appears as a threshold-governed mech- first, GFs establish a competence state, which is complemented anism underlying the decision to cross the R point.

524 Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

RESULTS enhanced upon the second pulse, compared to the first pulse. This difference in amplitude, as well as in the length of induction, Two Pulses of EGF Commit Mammary Epithelial Cells was independently verified by immunostaining of active ERK in to Proliferation the nucleus (Figure S2C). To explore commitment to proliferation, we employed clone To test whether enhanced ERK activation after the second 184A1 of normal human mammary epithelial cells (Hammond pulse is essential for proliferation, we applied a MEK inhibitor, et al., 1984). These cells were activated with EGF according to U0126. High concentration of U0126 (5 mM) strongly inhibited a protocol developed for fibroblasts (Jones and Kazlauskas, ERK activation, whereas lower concentrations resulted in pro- 2001): First, they were starved for GFs (16 hr) and then stimulated portionally reduced effects (Figure S2D). As shown in Figure 2D, for 1 hr with EGF, washed, and incubated in starvation medium when the second pulse of ERK was reduced by 50% (0.1 mM for 7 hr. Subsequent exposure to a second 1 hr pulse initiated U0126), entry into S phase was essentially abrogated, despite DNA synthesis 3 hr later (Figure 1A). This was confirmed by the remaining 50% increase in ERK phosphorylation. In contrast, multiple repetitions of the experiment, which were averaged in cells treated with a lower concentration of U0126 (0.05 mM), and presented in Figure S1A (available online) without normaliza- where ERK activation reached 80% of its uninhibited level, cells tion. In contrast, cells treated with a single pulse, or with two were able to enter S phase (Figure 2D and Figure S2D). Taken pulses separated by a shorter interval, displayed no comparable together, our observations indicated that the relative amplitude DNA synthesis (Figure S1B). Importantly, the two-pulse protocol and longer duration of ERK activation during the second pulse and the more conventional continuous exposure procedure are critical for S phase commitment. Interestingly, these results similarly impacted the capacity of cells to enter S phase (Fig- propose that ERK activation sets a threshold mechanism able ure 1B). A time-course analysis confirmed progressively higher to convert a graded input to an all-or-none output (digitization), BrdU incorporation signals and also indicated that the onset of required for S phase entry decision (Pardee, 1989). DNA synthesis occurs 12 hr after stimulation (Figure S1C), in The increase in phosphorylation of AKT and S6 is compatible line with a previous study performed with these cells (Stampfer with activation of the PI3K cascade. To determine the impor- et al., 1993). To focus on events regulating S phase entry and tance of this pathway in inducing S phase entry, we utilized avoid later effects, we adopted the 9–12 hr time window for the dual PI3K/mTOR inhibitor, LY294002. Figure 2E shows that measuring BrdU incorporation. inhibition of PI3K/mTOR, specifically at the second pulse, was To verify cell-cycle completion after the two-pulse scenario, sufficient for inhibition of S phase entry, whereas inhibition of we employed a methyl violet staining protocol, which is highly PI3K during the first pulse was ineffective (Figure S2E). This is reproducible when applied to strongly adherent cells. This assay in accordance with the conclusion that the second pulse may validated that two appropriately timed pulses promoted cell be substituted by direct activation of PI3K in cells expressing proliferation, whereas a single pulse was insufficient (Figure 1C). c-MYC and an active MEK (Jones and Kazlauskas, 2001). Parallel counting of DAPI-stained nuclei ensured that the differences measured by methyl violet staining were due to an Distinct Programs of Gene Expression Are Induced increase in cell number (Figure S1D). c-MYC and E2F coordinate by Each Pulse of EGF S phase entry, and they are regulated at the level of protein abun- Application of actinomycin D and cyclohexamide verified that de dance (c-MYC) and pRb phosphorylation (E2F) (Chen et al., novo transcription and translation are required during the late 2009; Meyer and Penn, 2008). Consistently, both parameters phase of EGF signaling, to achieve S phase entry (Figure S3A). were enhanced at the time of S phase entry in cells treated Hence, we employed Affymetrix GeneSet arrays at 17 time with two pulses, relative to cells treated with a single pulse, or points to determine dynamic changes in the abundance of all with two pulses separated by a shorter interval (Figure 1D messenger RNAs (mRNAs) altered by EGF (Figure 3A). A set of and Figure S1E). In conclusion, the observed increase in BrdU logical rules was applied to categorize genes into groups sharing incorporation, cell proliferation, c-MYC expression, and pRb expression profiles (see the Experimental Procedures). This phosphorylation confirm that 184A1 cells enter S phase upon identified ten profiles, including patterns representing genes two pulses of EGF, but not upon a single pulse. transiently inducted by either pulse, profiles exhibiting persistent or interval-limited changes, and a group of genes downregulated Subtly Different Phosphorylation Signals Are Induced by the second pulse (Table S2 and Figures S3B–S3K). by Each Pulse of EGF Since the outcomes of the two pulses differ remarkably, we The First EGF Pulse Is Sufficient for the Induction considered differences in the phosphorylation cascades out- of Metabolic Enzymes Essential for S Phase Entry lined in Figure 2A. To characterize activation patterns, we Interestingly, a profile of 35 persistently induced genes, whose analyzed cell extracts by reverse-phase protein array (RPPA) ascending abundance was not affected by a second pulse, dis- using antibodies to phosphorylated sites of key proteins (Fig- played enrichment (q value < 0.001) for enzymes associated ure 2B and Table S1). To substantiate the RPPA results, we per- with steroid, cholesterol, and lipid metabolism (Figure 3B, Fig- formed immunoblot analyses of ERK, AKT, and ribosomal ure S3B, and Table S3). Elevated expression of two of these protein S6, which confirmed that phosphorylation of the three genes, isopentenyl-diphosphate delta isomerase 1 (IDI1), and proteins was markedly increased by each pulse (Figure 2C and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), was vali- Figures S2A and S2B). Importantly, ERK phosphorylation was dated by real-time quantitative PCR (RT-qPCR; Figure S3L). the only measured event that was more prolonged and Notably, the early induction of S6 phosphorylation, a crucial

Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. 525 Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

A Normalized BrdU incorporation Starvation (S) 011.25 EGF (E) BrdU labeling 1S-7S-1S

1E-7S-1S p=0.0074 p=0.035 1E-7S-1E p=0.011 1S-7S-1E

Time (hours) 0681224 10

B Normalized BrdU incorporation 0 11.21.4 1.6 1S-7S-1S p=0.045 1E-7S-1E p=0.0045 p=0.18 1E-7E-1E

Time (hours) 0681224 10

C Normalized occupied area 011.2

1S-7S-1S p=0.00059 1E-7S-1E p=0.024 1E-7S-1S

Time (hours) 0682624 1S-7S-1S 1E-7S-1E 1E-7S-1S

Fold phosphorylation Fol (normalized (normalized expression D 0606060 60 60 60 60 60 60 60 60 st dt 1 EGF (min) tot=0) ot=0 1h 4h 5h 7h 7h 7h 7h 7h 7h 7h Starve interval ) 0.5 1 321 60 60 - - - nd 60 2 EGF (min) I 1h 3h 1h 2h 4h Starve interval 1E-7S-1E 112 ppRb (s780) 1.00 0.84 0.94 1.05 1.01 1.31 1.30 1.26 1.23 1.19 1.05 0.74 Fold phosphorylation II

58 c-MYC 1E-7S-1S 1.00 1.18 3.44 3.60 1.96 2.27 3.16 3.86 3.94 2.51 1.97 1.72 Fold expression III 45 Actin ppRb (S780) c-MYC

Figure 1. Human Mammary Epithelial Cells Commit to Proliferation upon Two Timed Pulses of EGF (A) 184A1 human mammary cells were GF starved for 16 hr. They were then either pulsed with EGF (‘‘1E,’’ red) for 1 hr or mock pulsed (‘‘1S,’’ green). Thereafter, cells were washed and incubated in starvation medium for 7 hr, as indicated, either followed by a second, 1 hr pulse of EGF or not. Cells were then washed and incubated for 3 hr with BrdU in starvation medium. Thereafter, the cells were ﬁxed, stained, and counted under a ﬂuorescent microscope. BrdU incorporation into DNA was measured by determining the ratio of BrdU- to DAPI-stained nuclei and was normalized according to the starvation control (1S-7S-1S). Error bars

526 Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

determinant of protein synthesis (Figure 2B), was consistent with Importantly, this was sufficient to prevent R point transition. In an increase in lipid biosynthetic processes upon the initial pulse. conclusion, EGR1, and possibly other TFs that exhibit the Hence, we speculated that early induction of metabolic 1st <2nd expression pattern, might participate in the digitization processes enables the increases in cell size and membrane of ERK signals. mass required for cell division. To test this, we pharmacologically To further address a model of mild EGR1 overshoot, we targeted two pathways: the AMPK pathway was activated with switched pulses between EGF and the insulin-like growth factor Metformin and AICAR, and Mevastatin was employed to inhibit 1 (IGF-1). Although IGF-1 could replace EGF as a first-pulse HMG-CoA reductase. Of relevance, metabolism inhibitors were inducer, the reciprocal order was ineffective (Figure S4D). Corre- previously shown to inhibit cell growth downstream to EGFR, spondingly, we observed enhanced EGR1 induction when cells primarily through inhibition of cholesterol and fatty acid synthesis were treated with IGF-1 and then with EGF, but no EGR1 (Guo et al., 2009). In the same vein, lipid metabolism genes are enhancement (or ERK activation) was observed with the inverse often upregulated in cancer models (Hirsch et al., 2010). Consis- combination (Figure 4D and Figure S4E). This observation further tently, incubation of cells with either inhibitor during the interval supported the model and motivated manipulation of the second completely abolished EGF-induced proliferation (Figure 3C), peak of EGR1. Cells were transfected with EGR1-specific supporting the hypothesis that increased lipid and cholesterol siRNAs immediately after the first pulse, which resulted in partial metabolism is essential for proliferation, despite being indepen- knockdown during the second pulse (Figure S4F). Importantly, dent of the second pulse. the observed reduction in EGR1 expression during the second pulse abrogated S phase entry (Figure 4E). In conclusion, the Differential Transient Induction of TFs by the Early enhancement of ERK activation instructs enhanced EGR1 induc- and Late Pulses tion at the second pulse, and this is required for S phase entry, in To focus on early transcriptional programs, we studied genes agreement with a threshold-setting mechanism that licenses that exhibited transient induction by both pulses and assigned crossing of the R point (Figure S4G). them to three profiles, according to the ratio of induction between the two pulses (Figures S3C–S3E). The immediate- The Second Pulse Downregulates p53-Controlled early TFs included in the profile denoted 1st <2nd (greater Antiproliferative Genes that Are Upregulated induction upon the second pulse; e.g., EGR1) and the profile by the First Pulse 1st >2nd (e.g., c-FOS) are shown in Figure 4A and Figure S4A, The expression profiles of one large group of EGF-induced respectively, along with verification of differential induction genes, denoted ‘‘downregulated by a second pulse,’’ implied (Figures S4B and S4C and Figure 4B). Interestingly, both induc- a restraining mechanism. Validation of the patterns of five genes tion of EGR1, an ERK-induced TF essential for mitogenic of this group with RT-qPCR confirmed the unique kinetics, in line responses, and the profile of ERK activation (Figure 2B) were with immunoblotting of one gene product, p27 (CDKN1B; enhanced at the second pulse. Hence, we hypothesized that Figures S5A and S5B). Interestingly, we noted that this profile the enhanced ERK signals are coupled to increased transcrip- contains well-characterized antiproliferative genes, seven of tion of genes of the 1st <2nd profile, and together they license which are known transcriptional targets of the p53 tumor R point transition. To examine this model, we related EGR1 to suppressor (Figure 5A). Hence, we focused on the possibility the aforementioned ability of U0126 to block R point transition that p53 might be involved in a restraining mechanism, which (see Figure 2D). As expected, complete ERK inhibition (U0126 is set up by the first pulse of EGF, but is removed by the second at 5 mM, see Figure S2D) essentially abolished EGR1 induction pulse. To test this model, we examined the downregulation of six (Figure 4C) and prevented cell proliferation (Figure 2D). In of the antiproliferative genes after treatment with the previously contrast, lower concentrations (0.05 mM), which reduced utilized combinations of EGF and IGF-1. As shown in Figure 5B, EGR1 expression by only 22%, did not interfere with prolifera- when cells were treated with two pulses of EGF, or with a first tion (Figure 2D). Application of an intermediate concentration pulse of IGF-1 followed by a second pulse of EGF, all six antipro- (0.1 mM) inhibited ERK activation by 54% and EGR1 transcrip- liferative genes we examined underwent more than 2-fold down- tion by 42%, thereby quenching extra activation of the pathway. regulation after the second pulse. In contrast, treatments that do represent standard errors calculated from at least 15 nonoverlapping photomicrograph fields (>500 nuclei). Significant p values of two-tailed student’s t test are indicated. The experiment was repeated three times. (B) 184A1 cells were GF starved as in (A) and then treated with two pulses of EGF or continuously stimulated with EGF for 9 hr. Cells were then washed and incubated for 3 hr with BrdU and fixed, and BrdU incorporation analyzed as in (A). Error bars represent standard errors calculated from at least 15 nonoverlapping photomicrograph fields (>500 nuclei). p values of two-tailed student’s t test are indicated. The experiment was repeated twice. (C) 184A1 cells were GF starved and treated as in (A). After the second pulse, cells were left in starvation medium for 17 hr, fixed, and stained with methyl violet. Cell-occupied area was then measured from four light photomicrographs of nonoverlapping fields. Error bars represent the standard errors calculated from triplicates. Significant p values of two-tailed student’s t test are indicated. Representative light photomicrographs are presented. The experiment was repeated twice. (D) Cells were GF starved and treated as in (A). At the indicated time points, cells were harvested and lysed, and cleared extracts were electroblotted. Phos- phorylation of Rb and abundance of c-MYC were determined (left) and quantified by densitometry. Signals were normalized to actin, and fold phosphorylation or expression was calculated (presented under each lane). The right panel presents the corresponding signals determined at the time of BrdU measurement (marked by Roman numbers). The experiment was repeated twice. See also Figure S1.

Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. 527 Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

A JNK JUN

MEK1 Erk1/2 p90RSK EGFR STAT3 TSC2 mTOR p70S6K S6

PI3K PDK1 AKT

LKB1 AMPK

ACC

B Fold C change 1st pulse 2nd pulse pERK1/2(T202/Y204) 1.27 pS6(S235/236) 0.69 01530606060 60 60 60 60 1st EGF (min) pS6(S240/244) 0.51 pSTAT3(Y705) 0.44 .5h 7h 7h 7h 7h 7h Starve interval pSTAT3(T727) nd cJUN 15 30 60 60 2 EGF (min) pACC(S79) .5h Starve interval pp70S6K(T389) pAKT(S473) pERK pLKB1(S428) 42 pAMPKa(T172) 1 6.54 4.73 2.42 1.55 0.57 6.58 8.99 7.54 1.34 Fold phosphorylation pp90RSK(T359/S363) 42 ERK 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Time (min): 2 4 60 9 80 4 2 0 2 40 0 6 70 00 2 120 1 2 360 4 480 5 5 5 57 6 6 72 54 5 6 6607 st 1st +2nd pulses 1 pulse only

Min Max

D Normalized BrdU incorporation % ERK activation U0126 0 11.21.4 U0126 (495 min) 1S-7S-1S - p=0.019 1E-7S-1E - 100

1S-7S-1S 0.1µM p=0.65 1E-7S-1E 0.1µM 46

1S-7S-1S 0.05µM p=0.0079 1E-7S-1E 0.05µM 80

Time (hours) 0681224 10

E Normalized BrdU incorporation LY294002 0 0.8 11.2 LY294002 1S-7S-1S - p=0.019 1E-7S-1E -

1S-7S-1S 50µM p=0.46 1E-7S-1E 50µM Time (hours) 0681224 10

Figure 2. Comparative Analyses of Phosphorylation Cascades Stimulated by Each EGF Pulse (A) A scheme presenting phosphorylation cascades activated by EGFR. Light blue-labeled proteins were analyzed with RPPA. (B) 184A1 cells were GF starved for 16 hr. Thereafter they were pulsed for 1 hr with EGF, washed, and incubated in starvation medium for 7 hr, followed by a second pulse with EGF (red) or no treatment (green). At the indicated time points, cells were harvested, and equal amounts of protein were used for RPPA analysis with the indicated antibodies. The mean of phosphorylation signals normalized to the respective total expression level (in triplicates) was calculated. The heat map presents the means in log2 scale, centered to the corresponding mean across all samples. The fold change in phosphorylation between the second pulse peak and the ﬁrst pulse peak is indicated (right column), if the difference between the peaks was signiﬁcant.

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B A IDI1 0 0 0 Time (min): 0 30 60 120 180 240 300 360 42 48 510540 600 66 720 C14orf1 ACSS2 HMGCR st nd CYP51A1 1 +2 pulses NM_000786.2 NSDHL Time (min): 0 30 60120 180 300 360 420480 510 540 600 660 510 540 600 660 1st pulse only 1st +2nd pulses 1st pulse only

C Min Max Normalized BrdU incorporation Inhibitor 0 1 1.4 Inhibitor - 1S-7S-1S p =0.037 - 1E-7S-1E

1S-7S-1S Metformin

1E-7S-1E Metformin 1S-7S-1S Mevastatin

1E-7S-1E Mevastatin 1S-7S-1S AICAR

1E-7S-1E AICAR Time (hours) 0681224 10

Figure 3. A Short Pulse of EGF Is Sufficient for the Induction of Metabolic Enzymes Essential for Cell Proliferation (A) A scheme depicting the setup of the microarray experiment. mRNA samples were isolated at the indicated time points (blue triangles). Red and green segments indicate EGF pulses and intervals, respectively. (B) 184A1 cells were treated as in Figure 1A. mRNA abundance was measured at the indicated time points with Affymetrix GeneSet microarrays. Shown are centered and normalized expression patterns of genes associated with cholesterol biosynthetic processes included in the ‘‘persistently induced’’ profile (see Figure S3B). (C) 184A1 cells were grown and processed as in Figure 1A except that cells were treated with Metformin (0.1 mM), Mevastatin (1 mM), or AICAR (0.5 mM) during the interval (shaded). Error bars represent standard errors calculated from at least 15 nonoverlapping photomicrograph fields (>500 nuclei). A significant p value of two-tailed student’s t test is indicated. The experiment was repeated three times. See also Figure S3 and Tables S2 and S3. not promote proliferation, such as a single EGF pulse or a combi- DNA synthesis (Jones et al., 1999; Kumar et al., 2006). In conclu- nation of EGF followed by IGF-1, elicited very modest downregu- sion, our results indicate that a late, PI3K-mediated process that lation of the six genes, consistent with restraining roles. downregulates a set of antiproliferation genes is necessary for S To identify signaling pathways mediating downregulation of phase entry. the group of antiproliferative genes, we used specific MEK Because the antiproliferative genes we identified undergo up- (U0126) and PI3K (LY294002) drugs. This revealed that activa- regulation at the first pulse and a fraction of the set is regulated tion of PI3K is necessary for downregulation of the set of six anti- by p53, we predicted that the early pulse of EGF activates p53. proliferative genes (Figure 5B), but U0126 did not affect the Of relevance, cell cycle-related pulses of p53 have recently been capacity of the second pulse to downregulate their expression detected (Loewer et al., 2010). To test this prediction, we (Figure S5C). In line with PI3K involvement, it has been reported isolated chromatin after EGF stimulation and measured the that a subset of apoptosis-promoting E2F1 target genes is fraction of DNA-bound p53. This uncovered early EGF-induced specifically repressed by PI3K signaling (Hallstrom et al., activation of p53’s transcriptional function, which persisted for 2008). Moreover, PDGF induces immediate as well as delayed 6–8 hr and decreased toward the end of the interval (Figure 5C). waves of PI3K activity, but only the later wave is obligatory for Importantly, upon a second pulse, p53 activation almost

(C) 184A1 cells were GF starved and treated as in (B). Cells were harvested at the indicated time points, lysed, and analyzed by immunoblotting. Quantiﬁed and normalized signals are presented under each lane. The experiment was repeated three times. (D) 184A1 cells were treated, and BrdU incorporation measured as in Figure 1A. Cells were treated with U0126 at the indicated concentrations 30 min prior to and throughout the second pulse (shaded area). Error bars represent standard error values (>500 nuclei). ERK activation was calculated according to Figure S2D. The experiment was repeated three times. (E) 184A1 cells were GF starved and treated, and BrdU incorporation measured as in (D). Cells were treated with LY294002 as indicated 30 min prior to and throughout the second pulse (shaded). Error bars represent standard error values. The experiment was repeated three times. See also Figure S2 and Table S1.

Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. 529 Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

A B 2nd vs. 1st peak EGR1 ratio CITED2 1 2 KLF10

0 0 0 0 0 Time (min): 30 60 0 0 6 120 18 3 360 420 480 510 540 6 6 c-FOS

Min Max EGR1

C U0126 (µM) -05- 0.5 0.1 .05 60 60 60 60 60 60 1st EGF (min) 7h 7h 7h 7h 7h 7h Starve interval 60 60 60 60 60 2nd EGF (min)

80 EGR1 % Induction: 100 30 47 58 78 46 Actin

D BrdU Ratio of fold EGR1 EGR1 incorporation induction (540min vs. 60min) pattern 1E-7S-1S - 0.12 1E-7S-1E + 1.8 1IGF-7S-1E + 2.82 1E-7S-1IGF - 0.189

E siRNA Normalized BrdU incorporation incubation 0 1 1.25 1S-7S-1S - p=0.032 1E-7S-1E

1S-7S-1S siControl p=0.032 1E-7S-1E

1S-7S-1S siEGR-1 p=0.64 1E-7S-1E

Time (hours) 0681224 10

Figure 4. Differential Induction of Immediate-Early Transcription Factors by the Two Pulses (A) 184A1 cells were treated with two pulses of EGF, as in Figure 3B. Shown are centered and normalized expression levels of the indicated immediate-early induced TFs from the proﬁle denoted 1st <2nd (Figure S3D). (B) 184A1 cells were treated with two pulses of EGF. At the end of each pulse, mRNA was isolated, followed by cDNA synthesis and RT-qPCR with primers for either c-FOS or EGR1. Presented are the average ratios of expression, calculated from four biological repeats. Error bars represent standard error values. (C) GF-starved 184A1 cells were pulsed for 1 hr with EGF, washed, and incubated for 7 hr in starvation medium, followed by a second EGF pulse. Cells were treated with U0126 at the indicated concentrations, 30 min prior to and throughout the second pulse. Immunoblotting was used to quantify EGR1 levels of induction relative to the level at 1E-7S-1E, normalized to actin. (D) GF-starved 184A1 cells were pulsed for 1 hr with EGF (‘‘1E,’’ red), IGF-1 (‘‘1IGF,’’ purple), or left untreated (‘‘1S,’’ green). After GF removal, cells were incubated in starvation medium for 7 hr, followed by a second pulse of EGF or IGF-1, as indicated. For BrdU incorporation analysis, see Figure S4D. For determination of EGR1 fold induction, mRNA was isolated at the end of each pulse (blue triangles), and cDNA was analyzed by RT-qPCR. The right column schematically presents time proﬁles of EGR1’s patterns of expression.

530 Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

disappeared (compare lanes 7 and 9 in Figure 5C). Thus, the sure to GFs, or two short pulses confined to a time window of pattern of p53 activation correlated with the kinetics of expres- approximately 9 hr (Jones and Kazlauskas, 2001). In this report, sion of its targets. Notably, the abundance of p53 in the soluble we applied the discontinuous stimulation protocol to human fraction exhibited only moderate changes on EGF treatment mammary epithelial cells and unraveled three transcriptional (Figure S5D). mechanisms that orchestrate entry into the S phase: (1) a persis- In an effort to firmly establish involvement of p53, we knocked tent increase in expression of genes associated with lipid biosyn- down its expression using specific small interfering RNAs thesis (initiated by the first EGF pulse), (2) a threshold mechanism (siRNAs). This treatment reduced p53 levels by 85% and resulted that requires enhanced activation of ERK and induction of EGR1 in 50%–70% lower expression of the set of p53 target genes by the second pulse, and (3) activation (by the second pulse) of (Figures S5E and S5F). We therefore examined whether a reduc- PI3K, which suppresses a set of antiproliferative genes. Several tion in p53 would bypass the need for a second pulse. Remark- of these antiproliferative genes are transcriptional targets of p53, ably, p53 knockdown enabled cells to enter S phase upon a single which is activated by the first pulse, safeguarding the system pulse of EGF, in contrast to control cells that required two pulses against proliferative responses to inconsistent, fortuitous GFs (Figure 5D and Figure S5G). Interestingly, comparison of p53 signaling. knockdown with control cells detected increased expression of EGR1, as long as 7 hr after the first EGF pulse (Figure S5H), sug- Early Induction of Metabolic Enzymes gesting a mechanism that sensitizes cells to proliferation upon In order to divide, cells require relatively large quantities of lipids, a single EGF pulse, once the p53-dependent restraining mecha- proteins, and nucleotides, which are used to increase cell size nism is abrogated. Next, we tested the prediction that PI3K and replicate DNA. Accordingly, it was shown that cellular activity is not needed during the second pulse, if p53 activity is growth rates are tightly coordinated with the length of the cell compromised. Indeed, inhibition of PI3K with LY294002 cycle, suggesting the existence of a size sensor at G1, which completely blocked S phase entry by control cells, but no effect maintains a roughly constant cell mass over many cycles of LY294002 was observed upon p53 knockdown (Figure S5I). (Dolznig et al., 2004). A recent study of the relationships between This result underscores the role of PI3K as a late G1 suppressor cell size and the cell cycle demonstrated an accelerative, of p53-regulated antiproliferative processes. size-dependent growth rate during G1, implying an intrinsic Since forced p53 reduction in epithelial cells preempted the size-regulating machinery (Tzur et al., 2009). Consistent with requirement for a second pulse, we asked whether the underlying these studies, three aspects of our experimental evidence indi- mechanism is generalizable and can be extended to the original cated an increase in metabolic processes after a single pulse fibroblast cell system, in which the two-pulse scenario was first of EGF: elevated S6 phosphorylation, which translates to accel- established. Congruent with the original report (Jones and Ka- erated protein synthesis (Figure 2), induction of genes associ- zlauskas, 2001), stimulation of NIH-3T3 cells, which express ated with lipid and sterol biosynthetic pathways (Figure 3B), wild-type p53 (Huang et al., 1996), with two pulses of PDGF signif- and inhibition of the two-pulse commitment to cell-cycle icantly increased their proliferation (Figure 5E). Although knock- engagement induced by metabolic inhibitors (Figure 3C). It is down of p53 was less efficient than in 184A1 cells, it enhanced important to note that although they are obligatory, the early growth of starved fibroblasts (Figures S5E and S5J). Importantly, metabolic events are not sufficient for cell proliferation, since forced reduction of p53 expression completely eliminated the a single pulse of EGF gave rise to their induction, whereas S necessity for a second pulse of PDGF to promote proliferation phase entry required an additional pulse. (Figure 5E), similar to the results obtained with 184A1 cells. Taken together, our results indicate that a single pulse of EGF An ERK/EGR1 Gating Mechanism initiates a priming process, which includes lipid metabolism and Because metabolic processes could not explain the gating drives cells through early G1, but at the same time it also induces mechanism involved in restriction point crossing, we focused a restraining process that prevents commitment to proliferation. on events that differ between the first and the second pulses of The latter involves activation of p53 and a set of antiproliferative EGF. Two activation indicators of the ERK pathway, namely genes. When cells are exposed to a second EGF pulse, signaling ERK phosphorylation and EGR1 abundance, displayed through PI3K/AKT releases the constraint. If enhanced activation enhanced signals after the second EGF pulse (Figures 2 and of ERK and consequent induction of EGR1 exceed a threshold 4). We further found that inhibition of the excessive activation and in parallel the antiproliferation genes are downregulated, of ERK or of the increased induction of EGR1 abolished cell cells will cross the R point and commit to proliferation (see model proliferation, which implied a threshold mechanism. Accord- in Figure 6). ingly, to cross the R point, the activation of ERK and downstream induction of EGR1 must reach the set threshold, thus ensuring DISCUSSION that weak or inconsistent signals would not trigger cell proliferation. Compatible with a late ERK-centered gating mechanism, it To irreversibly cross the restriction point (R) of the cell cycle, has previously been reported that the pattern of ERK activation fibroblasts have been shown to require either continuous expo- upon continuous exposure of fibroblasts to GFs is biphasic

(E) GF-starved 184A1 cells were treated and analyzed as in Figure 1A. The shaded rectangle indicates transfection with control or EGR1-speciﬁc siRNA oligonucleotides. Error bars represent standard error values. p values of two-tailed Student’s t test are indicated. See also Figure S4.

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A B Fold change (2nd pulse, 600min vs. 480min) BTG2 SESN1 BrdU SESN1 BTG2 FBXW7 BCL6 CDKN1B SESN2 SESN2 Stimulations incorporation BCL6 1E-7S-1S 1.38 1.01 0.94 1.18 1.27 1.09 TP53INP1 - CDKN1B 1E-7S-1E + 0.45 0.31 0.46 0.32 0.33 0.46 TP53TG1 FBXW7 1IGF-7S-1E + 0.41 0.24 0.43 0.25 0.37 0.21 CDKN2C GAS2L3 1E-7S-1IGF - 0.78 0.59 0.68 0.84 1.03 0.85

0 0 0 0 0 0 0 0 0 Time (min): 30 60 80 0 60 20 80 10 4 6 4 0 6 120 1 3 3 4 4 5 5 60 6 51 5 6 6 1E-7S-1E 0.84 1.35 1.77 1.10 1.04 1.01 LY294002 - Min Max D Normalized BrdU incorporation 0 1 1.5 C 1S-7S-1S p 0606060 60 60 60 60 60 60 1st EGF (min) =0.013 - 1E-7S-1E 1h 3h 5h 7h 7h 7h 7h 7h Starve interval 60 60 - - 2nd EGF (min) 1E-7S-1S 1h 1h 2h Starve interval 1S-7S-1S p 50 p53 =0.072 1.00 2.51 2.97 3.56 2.98 1.39 0.66 0.50 1.67 0.93 Fold expression siControl 1E-7S-1E 32 Histone1 1E-7S-1S 1 2 3 456 78910 1S-7S-1S p=0.0075 sip53 1E-7S-1E p=0.036 1E-7S-1S E Normalized occupied area 2 4 6 Time (hours) 0681224 10 1S-7S-1S siControl 1P-7S-1S siControl p=0.0001 1P-7S-1P 1S-7S-1S sip53 sip53 1P-7S-1S p=0.211 1P-7S-1P 1S-7S-1S 1P-7S-1S 1P-7S-1P Time (hours) 0682724

Figure 5. The Second Pulse Downregulates Antiproliferative Genes Induced by the First Pulse (A) 184A1 cells were treated with two pulses of EGF. Shown are centered and normalized levels of antiproliferation genes (marked in black are known p53 targets) from the profile ‘‘downregulated by a second pulse’’ (see Figure S3F). (B) 184A1 cells were treated as in Figure 4D. Where indicated, cells were treated for 30 min with LY294002. BrdU incorporation results are presented in Figure 2E and Figure S4D. For determination of fold change, mRNA was isolated before the second pulse and 60 min after completion of the pulse (blue triangles), and cDNA was analyzed by RT-qPCR. Listed are the ratios of expression levels after and before the second pulse. (C) GF-starved cells were pulsed for 60 min with EGF, washed, and incubated in starvation medium for 7 hr, followed by a second pulse. At the indicated time points, cells were harvested for a chromatin association assay, and DNA-bound proteins were isolated and analyzed by immunoblotting. (D) Cells were transfected with control or p53-specific siRNAs. Twenty-four hours later, the cells were replated on coverslips, and another 24 hr later, they were GF starved for 16 hr and treated with EGF, and BrdU incorporation was measured. Error bars represent standard errors calculated from 15 nonoverlapping photomicrograph fields. p values of two-tailed student’s t test are indicated. (E) NIH-3T3 cells were transfected with control or p53-specific siRNAs. Twenty-four hours later, the cells were replated, and another 24 hr later they were GF starved for 24 hr, treated for 1 hr without (‘‘1S,’’ green) or with (‘‘1P,’’ blue) PDGF, washed, and incubated in starvation medium for 7 hr, followed by a second PDGF pulse. Cells were left in starvation medium or PDGF-containing medium for additional 18 hr and were then fixed and stained with methyl violet. Repre- sentative photomicrographs are shown (right). The cell-occupied area was measured from five photomicrographs (left). Error bars represent standard errors of triplicates. The experiment was repeated twice. p values of two-tailed Student’s t test are indicated. See also Figure S5.

(Meloche et al., 1992). Further, the late phase of ERK activation, newly synthesized following EGF stimulation to feedback control which occurs at mid-G1, is essential for S phase entry, and its downstream signals (Amit et al., 2007). inhibition prevents cell proliferation (Sah et al., 2002). Our study leaves open mechanisms underlying the second pulse enhance- A p53-Mediated Mechanism Dampens Mitogenic Noise ment of the ERK-EGR1 signals. One potential mechanism The transcriptional response to EGF is very complex, yet a involves ERK-speciﬁc dual-speciﬁcity phosphatases, which are portion of the response could be grouped into ten temporal

532 Molecular Cell 42, 524–535, May 20, 2011 ª2011 Elsevier Inc. Molecular Cell EGR1 and p53 Regulate Commitment to Proliferation

Figure 6. Schematic Representations of the Proposed Biochemical Events Elicited by a Single and a Dual EGF Pulse The ﬁrst pulse of EGF induces expression of lipid biosynthesis-associated genes, along with activation of p53. The latter propels expression of antiproliferation genes, such as BTG2 and SESN1. When cells are treated with a second pulse of EGF, enhanced activation of ERK and subsequent induction of EGR1 exceed a critical threshold. In parallel, signaling through PI3K and the resulting suppressed expression of antiproliferative genes permit cells to cross the restriction point (R) and enter the S phase. See also Figure S6.

The three transcription-based regula- tory mechanisms of S phase entry we unveiled open interesting questions for future research. For example, p53 and PI3K are assembled into an incoherent feed-forward process, which has the patterns (Figure S3). One group, which includes multiple anti- characteristics of a checkpoint allowing transition to the next proliferative genes, was strongly suppressed by the second step only when sufficient GF signals are provided (Figure S6). pulse of EGF (Figure 5). Suppression of antiproliferative genes On the other hand, p53 is often deleted and PI3K is frequently during G1 has previously been shown in quiescent fibroblasts mutated in human cancers, raising the intriguing possibility that 6–12 hr after serum stimulation (Iyer et al., 1999) or upon sus- the aberrant p53/PI3K checkpoint sensitizes many tumors to tained activation of ERK (Yamamoto et al., 2006). Interestingly, sporadically available GFs. Mutations of EGFR, which induce several genes included in this group have previously been char- basally weak activation, are similarly frequent in cancer, reinforc- acterized as targets of p53. Congruent with p53 involvement, ing potential clinical implications of the feed-forward process. the first pulse of EGF increased the tight association of p53 The gating mechanism involving late enhancement of the ERK- with chromatin, which persisted for 6–8 hr (Figure 5C). It is EGR1 module raises additional questions. While it is likely that worth noting that excessive mitogenic signals involving RAS this module digitizes graded GF signals, thereby blocking induc- and c-MYC similarly engaged the antiproliferative activity of tion of S phase entry by inconsistent pulses of GFs, molecular p53 (Zindy et al., 1998). To confirm a role for p53, we knocked mechanisms that generate enhanced late signals and compute down its expression. As expected, removal of p53 enabled both fold activation are matters for future investigation. epithelial and fibroblastic cells to enter S phase upon a single pulse of EGF, in contrast to the two-pulse requirement for EXPERIMENTAL PROCEDURES normal cells. These observations indicate that p53 protects cells from excessive or untimely responses by imposing a Cell Lines and siRNA Transfection constraining mechanism, which is relieved by a second GF Human mammary 184A1 cells were maintained in DFCI medium (Band and Sager, 1989). NIH-3T3 cells were grown in DME medium supplemented with stimulation. In line with this, it has previously been reported 1 mM sodium pyruvate. For further details, see the Supplemental Experimental that lung carcinomas that harbor EGFR mutations also carry Procedures. siRNA transfections were carried out with HiPerFect (184A1 cells; p53 mutations (Mounawar et al., 2007), which is likely to QIAGEN, Germany) or Dharmafect1 (NIH-3T3; Lafayette, CO). unleash full transformation. In addition, the efficacy of treating hepatocellular carcinoma with an EGFR-blocking antibody, Ce- BrdU Incorporation Assay tuximab, displayed dependence on wild-type p53 (Huether Incubation with the BrdU labeling reagent was carried out for 3 hr, followed by et al., 2005). fixation and staining with a BrdU detection kit (Roche Diagnostics GmbH, Germany). Cells were visualized with a Nikon Eclipse 90i microscope, and The PI3K/AKT pathway contributes to cell survival and photos were captured with the Image-Pro software. BrdU- and DAPI-stained suppresses the expression of antiproliferative genes, such as nuclei were counted from at least 15 fields of each treatment. CDKN1B/p27, thus shifting the balance toward proliferation after activation of E2F (Hallstrom et al., 2008; Sa and Stacey, 2004). Methyl Violet Staining Furthermore, activation of AKT reduced hypoxia-induced Cells were washed with ice-cold saline, fixed in methanol, incubated with 0.3% transcriptional activation of p53 (Yamaguchi et al., 2001) and methyl violet for 5 min at À20 C, and washed. Pictures from four nonoverlapping delayed the onset of p53-mediated apoptosis (Sabbatini and fields were collected with a Leica binocular. The cell-occupied area was determined with Cell Profiler (http://www.cellprofiler.org/)(Carpenter et al., 2006). McCormick, 1999). Accordingly, we demonstrate here that inhibition of PI3K during the second pulse of EGF abrogated Reverse-Phase Protein Array Analysis suppression of the anti-proliferative group of genes, and pre- Cell pellets were lysed in RPPA buffer (1% Triton X-100, 50 mM HEPES vented proliferation. [pH 7.4], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM

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Na-pyrophosphate, 1 mM Na3VO4, 10% glycerol, and protease and phospha- ACKNOWLEDGMENTS tase inhibitors [Roche Diagnostics GmbH, Germany]) for 20 min on ice. After centrifugation, protein concentration was assayed with the BCA reagent The authors would like to thank Sara Lavi, Doron Ginsberg, Noa Bossel, Ido (Pierce, Rockford, IL), and then 4XPSB (40% glycerol, 8% SDS, 0.25 M Amit, and Ami Citri for their help and Eylon Shahar for technical support. Our Tris-HCl [pH 6.8], and 10% 2-mercaptoehtanol) was added to the cleared research is supported by grants from the National Cancer Institute (including lysates followed by boiling. Samples were serially diluted, spotted onto 4R37CA072981, Cancer Center Support Grant, and grant P30 CA16672), nitrocellulose-coated slides, and probed with antibodies (Supplemental the European Commission, the German-Israeli Project Cooperation, the Israel Experimental Procedures), and signals were quantified as previously Cancer Research Fund, the Dr. Miriam and Sheldon G. Adelson Medical described (Amit et al., 2007). Research Foundation, the Kekst Family Institute for Medical Genetics, and the M.D. Moross Institute for Cancer Research. Y.Y. is the incumbent of the Harold and Zelda Goldenberg Professorial Chair and E.D. of the Henry Immunofluorescence Staining J. Leir Professorial Chair. Cells were grown on coverslips and treated as indicated. Cells were then fixed in paraformaldehyde (3%) for 15 min and stained with anti-pERK antibody Received: October 10, 2010 followed by Cy2-conjugated anti-mouse secondary antibody. Nuclei Revised: February 20, 2011 were stained with DAPI during the last washing step. See the Supplemental Accepted: April 16, 2011 Experimental Procedures for details regarding visualization and intensity Published: May 19, 2011 measurement.

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